Asian Journal of Medical Sciences 3(1): 1-7, 2011
ISSN: 2040-8773
© Maxwell Scientific Organization, 2011
Received: September 30, 2009
Accepted: January 28, 2010
Published: February 25, 2011
Effect of Fructose and Glucose Co-Administration on Ethanol- Induced
Changes in Lipids and Liver Morphology
1
I. Onyesom and 2V.J. Ekanem
1
Department of Medical Biochemistry, Delta State University, Abraka, Nigeria
2
Department of Morbid Anatomy, University of Benin, Benin City, Nigeria
Abstract: This study investigates the influence of oral fructose on Blood Ethanol Elimination Rate (BEER)
and the effect of glucose and fructose co-administration on ethanol-induced changes in blood/tissue lipids and
hepatic microstructure in experimental rabbits. Thirty-five male adult New Zealand white rabbits were
purchased and separated into five experimental groups (n = 7 per group). The groups were given orange juice
(control group), ethanol, ethanol + glucose, ethanol + fructose, and ethanol + glucose and fructose, once daily
for 15 weeks. Blood samples were analyzed for lipids at 0 (basal), 5th, 10th and 15th week of exposure. BEER
was however, determined after seven days of allowing the animals to acclimatize to feed and the animal house
environment. Tissue lipids and hepatic microstrcture were examined at the end of the 15-week exposure.
Standard reagents, instruments and procedures were used at the different stages of the experiment. Results show
that fructose and fructose + glucose administrations significantly (p<0.05) increased BEER by 46.1% and
50.6%. Fructose + glucose co-administration progressively increased the blood triacylglycerol (TAG) levels
in ethanol fed rabbits at the 5th (0.68±0.03 mmol/L), 10th (0.75±0.07 mmol/L; p<0.05) and 15th (0.92±0.04
mmol/L; p<0.05) week of exposure when compared with the basal (0 week) value (0.56±0.02 mmol/L). TAG
also accumulated in the livers of fructose + glucose co-treated animals (1.86±0.031 mmol/L; p<0.05; control:
0.362±0.016 mmol/L) at the end of the 15 week treatment period. The hepatic microstructures for ethanol,
ethanol + fructose, and ethanol + glucose and fructose - treated rabbits showed evidence of fatty hepatitis. The
hypothesis of adding glucose to the administration of fructose in rabbits receiving ethanol treatment is
ineffective because it increases the liver tissue levels of TAG and affects the healthiness of the animals.
Key Words: Ethanol, fibrosis, fructose, glucose, hepatitis, liver, triacylglycerol
effective therapeutic dose (Berman et al., 2003) has been
recognized to induce gastric distress (due to
malabsorption) and increase blood lipids, but when given
in combination with glucose, fructose is known to be well
absorbed (Truswell et al., 1988). Whether the coadministration of fructose and glucose also ameliorates
the fructose-induced modification in blood lipid and tissue
levels is not clearly established.
Thus, this study attempts to verify the effect of oral
fructose on Blood Ethanol Elimination Rate (BEER) and
report the influence of glucose and fructose coadministration on BEER and ethanol-induced changes in
blood/tissue lipid contents and associated alternations in
the microstructure of the liver - the main organ for ethanol
metabolism.
INTRODUCTION
Extensive and prolonged ingestion of alcohol is
injurious to health, alcohol abuse and addiction are
growing, and constitute serious problems in many
communities including ours. Not withstanding, the
management of alcoholism and allied diseases has
recorded little success.
Reports available (Masters, 2007) indicate that the
use of naltrexone, disulfiram, acamprosate, topiramate
and ondansetron, and recently, methylene blue
(Vonlanthen et al., 2000) has not yielded convincing
results in humans. It is on this note that the treatment
value of some supportive agents that could enhance blood
alcohol clearance are currently being investigated.
There are accumulated reports that fructose is capable
of accelerating the disappearance of alcohol from the
bloodstream (Mascord et al., 1992; Berman et al., 2003)
and so, could be beneficial in the treatment of alcohol
intoxication. However, oral administration of fructose
alone in solution at amounts exceeding 1 g/kg - the
MATERIALS AND METHODS
Study laboratory and period: The study was conducted
in the Alcohol Bioresearch Laboratory, Department of
Medical Biochemistry, Delta State University, Abraka,
Nigeria, between August and December, 2008.
Corresponding Author: Dr. I. Onyesom, Department of Medical Biochemistry, Delta State University, Abraka, Nigeria
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Asian J. Med. Sci., 3(1): 1-7, 2011
and weekly weights were determined and recorded. From
these records, feed consumption per rabbit per week and
weekly weight changes were obtained. The dose and
research design were based on previous documents
(Onyesom and Anosike, 2005). The animal care complied
with the guideline of the National Institutes of Health
(NRC, 1985).
Animals: Thirty-five male adult New Zealand white
rabbits with an initial mean weight of 1.45±0.12 kg, were
purchased from Edo Agricultural Development
Programme (ADP) Farm in Benin City. Growers’ mash
was purchased from Bendel Feed and Flour Mills
(BFFM), Ewu, Edo State. The animals were housed in
metal hutches and had free access to food and water for
one week to acclimatize them to the feed and laboratory
environment. Thereafter, they were divided into five
different groups, with seven animals.
Blood sample collection: Fasting whole blood was drawn
from each rabbit’s ear vein under anaesthesia with
intraperitoneal injection (50 mg/kg pentobarbital, Sanofi
Sante - Animale), at 0 (baseline) and at the 5th, 10th and
15th week of treatment into lithium-heparinized tubes
using sterile disposable, 21-guage hypodermic needles
and syringes. The whole blood sample was centrifuged at
1200 x g for 5 min at room temperature to separate the
plasma which was then removed, stored frozen and
analyzed within 48 h.
Treatment: Each group was labelled according to the
treatment as: Group OJ (control: orange juice-treated
rabbits), Group E (ethanol-treated rabbits), group EG
(ethanol + glucose-treated rabbits), group EF(ethanol +
fructose-treated rabbits) and group EG/F (ethanol +
glucose and fructose co-treated rabbits).
Group OJ animals were given orange juice, group E
animals received 1.5 g ethanol/kg body weight (diluted to
20% with orange juice plus 1.5 g normal saline/kg) as
single daily dose. Group EG also received the ethanol
dose, but was given in addition, 1.0 g glucose and 0.5 g
normal saline/kg body weight after about 20 min of the
ethanol dose. The treatment of group EF animals was
similar to those in group EG except fructose in lieu of
glucose. Then, Group EG/F animals also received the
ethanol dose but were in addition given 1.0 and 0.5 g of
fructose and glucose/kg body weight. The dosing regimen
was based on the experience with fructose in the treatment
of alcohol intoxication (Berman et al., 2003). Animals in
the various groups were treated as described along with
their feeds for a continuous period of 15 weeks.
On the first day of feeding, Blood Alcohol Levels
(BAL) were determined by the enzymatic-colorimetric
method (Bucher and Redetzki, 1951) every 20 min for 2
h using whole blood collected from the ear vein of each
rabbit. Whole blood samples for BAL assay were
collected at about 11:00 am and this time was 4 h after
breakfast. The BAL obtained were used to plot the blood
alcohol-time curve for each rabbit and from such curves,
the time taken to return to zero BAL (intersect on x-axis)
was estimated. Blood Ethanol Elimination Rate (BEER)
was then derived by dividing the alcohol dose (150
mg/kg) with the time (h) at the intersect for each rabbit.
The values derived from all the rabbits in a group were
used to calculate the mean±SD.
Preparation of tissues for biochemical study: At the
end of the 15-week treatment period, five rabbits from
each group were sacrificed under anaesthesia by cervical
dislocation and their livers, brains and hearts were quickly
excised and rinsed in cold normal saline (0.9% NaCl
solution). Then half gramme of each tissue was
homogenized with 5.0 mL distilled water and the
homogenate was centrifuged (1,500 x g for 7 min) to
separate supernatant from tissue delbris. The supernatant
was collected into bijou bottle, stored frozen and analysed
within 48 h.
Assay methods: Triacylglycerol (TAG) levels in plasma
and tissue extracts (supernatant) were estimated using the
enzymatic-endpoint colorimetric method as described
(Chawla, 1999). Total cholesterol values in both plasma
and tissue extracts were assayed as previously described
(Allain et al., 1974). Colorimetric method (Burstein and
Mortin, 1969) was used to determine the amounts of
cholesterol in HDL fraction for both plasma and tissue
extracts. LDL-cholesterol was mathematically estimated
(Friedewald et al., 1972).
Histopathological examination: The excised liver tissue
obtained from the remaining two rabbits in a group were
immediately fixed in 10% formol saline, and then
processed for light microscopy at the Department of
Morbid Anatomy, University of Benin Teaching Hospital,
Benin City, Edo State, Nigeria, using the hematoxylin and
eosin (H and E) stains. The developed slides were
examined and observations were recorded. The liver was
taken because it is the tissue most vulnerable to ethanol
toxicity.
Feeding: Each animal in each group was presented with
40 g feed twice daily. Clean drinking water was liberally
provided and the hutches were cleaned regularly. Feeds
were mixed with water in a ratio of 10:1 (w/v) so as to
achieve a texture acceptable to the animals. Stale feed
remnants were weighed and regularly discarded. The
feeding experiment was conducted at room temperature
(about 29ºC) in 12-h day light cycle. Daily feed remnants
Statistical analysis: The data are presented as
mean±SD. Statistical significance of the differences
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Asian J. Med. Sci., 3(1): 1-7, 2011
Table 1: Effect of fructose and glucose co-administration on Blood
Ethanol Elimination Rate (BEER) in Albino rabbits
Treatment
BEER (mg/kg/h)
Ethanol
219±6
(1.5 g [20%]/kg: n = 7)
Ethanol + Glucose
221±8 (0.9)
(1.5 g[20%]/kg + 1.0 g/kg: n = 7)
Ethanol + Fructose
320±11*(46.1)
(1.5 g[20%)]/kg + 1.0 g/kg: n = 7)
Ethanol + glucose and fructose
330±9* (50.6)
(1.5 g[20%]/kg + 0.5 and 1.0 g/kg: n = 7)
Values are expressed as Mean ± SD for n = 7 rabbits per treatment
group; *: Significantly different from the BEER value for ethanol alone;
Values in parenthesis are percentages increases in BEER values
compared with the BEER value for ethanol alone
From Table 2, it can be observed that ethanol
administration progressively increased plasma TAG levels
and these were further increased by the inclusion of either
fructose or glucose + fructose. The values obtained when
ethanol + glucose were co-administered were similar in
pattern to the comparable control values, though
proportionately higher. These differences were not
significant when compared with the basal (0 wk) or
comparable control value (p>0.05).
The changes in total cholesterol induced by the
different treatments were similar and none differed
significantly (p>0.05) from the basal or comparable mean
Table 2: Changes in mean plasma lipids induced by different treatments in experimental rabbits
Duration of treatment (wk)
-------------------------------------------------------------------------------------------------------0
5
10
15
-------------------------------------------------------------------------------------------------------Analyte
Treatment
Changes in plasma lipid values (mmoL/L)
Triacylglycerol
Control
0.52±0.02
0.54±0.12
0.55±0.04
0.54±0.02
Ethanol
0.61±0.02
0.65±0.04
0.71±0.02*
0.77±0.02*
Ethanol + glucose
0.53±0.01
0.57±0.01
0.63±0.02
0.66±0.03
Ethanol + fructose
0.63±0.09
0.72±0.02
0.76±0.02*
0.81±0.09*
Ethanol + glucose & fructose
0.56±0.02
0.68±0.03
0.75±0.07*
0.92±0.04*
Total cholesterol
Control
2.15±0.31
2.80±0.44
2.00±0.32
1.80±0.08
Ethanol
2.55±0.08
1.90±0.37
2.85±0.37
2.70±0.11
Ethanol + glucose
2.00±0.04
2.70±0.02
2.25±0.19
2.10±0.03
Ethanol + fructose
1.85±0.07
2.00±0.02
1.82±0.08
2.65±0.07
Ethanol + glucose & fructose
2.15±0.08
3.35±0.03
3.65±0.61
3.40±0.06
HDL-cholesterol
Control
0.86±0.23
0.76±0.04
0.82±0.11
0.80±0.23
Ethanol
0.98±0.35
1.06±0.11
1.19±0.04
1.20±0.04
Ethanol + glucose
0.78±0.82
0.86±0.04
0.96±0.01
0.94±0.05
Ethanol + fructose
0.92±0.07
0.98±0.01
1.10±0.03
1.12±0.61
Ethanol + glucose & fructose
0.71±0.06
0.92±0.01
1.07±0.04
1.06±0.37
LDL-cholesterol
Control
1.04±0.08
0.90±0.40
0.98±0.19
0.76±0.60
Ethanol
1.09±0.11
0.98±0.19
1.34±0.40*
1.15±0.22*
Ethanol + glucose
0.98±0.01
0.88±0.18
1.01±0.02
0.60±0.14
Ethanol + fructose
0.64±0.18
0.85±0.05
0.38±0.02
1.16±0.1*
Ethanol + glucose & fructose
0.85±0.37
1.12±0.55
2.14±0.54*
1.92±0.26*
Data are expressed as the mean ± SD of determination on seven rabbits per group; *: Significantly different from basal (0 week) value at p<0.05
reflection of the changes in the cholesterol contents of the
lipoprotein fractions. At the end of the 15-week exposure
period, ethanol treatment increased the level of HDLcholesterol the most, followed by ethanol + fructose
treatment and then, ethanol + glucose and fructose, but the
effect of these treatments on LDL-cholesterol content
were undulating, and the changes produced by ethanol
and ethanol + glucose and fructose were significant
(p<0.05) at the 10th and 15th week of administration.
Even though, ethanol-induced increase in HDL has
been reported to be cardio-protective (Lau et al., 1995) in
the absence of other aetiologic risk factors, the increase in
LDL-cholesterol for the ethanol + glucose and fructosetreated animals could enhance the possibility of
atherosclerosis since increase in this lipoprotein fraction
has been reported to contribute to the formation of fatty
streaks in arteries (Gaziano et al., 1993).
Table 3 shows that changes in total cholesterol
obtained from the different treatments did not
significantly (p>0.05) differ among the tissues, but
between treatment groups was assessed by Student’s
t-test. The time-courses of blood (plasma) lipids were
analysed by repeat measure analysis of variance
(ANOVA) followed by Dunnett’s test for multiple
comparisons and statistical significant difference was
established at p<0.05. EPI computer software was used.
RESULTS
The data obtained are shown in Table 1-4, while
Fig. 1-5 are the histopathological reports of the hepatic
tissues examined.
Table 1 shows the effect of fructose and glucose coadministration on Blood Ethanol Elimination Rate
(BEER). Evidence indicates that fructose significantly
increased BEER (p<0.05) by 46.1%, and the coadministration of glucose and fructose further increased
the BEER value to 50.6% (p<0.05). But glucose had no
significant (p>0.05) influence on BEER value as it only
increased it by 0.9%. These changes seem to be a
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Asian J. Med. Sci., 3(1): 1-7, 2011
Table 3: Level of tissue lipids at the end of the 15-week treatment period.
Tissue lipids (mmol/L)
------------------------------------------------------------------------------------------------------------------------------------------------Liver
Brain
Heart
------------------------------------------------------------------------------------------------------------------Treatment
TAG
T. chol.
TAG
T. chol.
TAG.
T. chol
Control
0.362± 0.016
0.622± 0.037
0.328± 0.021
0.932± 0.018
4.165± 0.230
0.518± 0.037
Ethanol
1.781± 0.036*
0.777± 0.055
0.396± 0.018
1.230± 0.064
0.940± 0.021*
0.544± 0.018
Ethanol + glucose
0.435± 0.034
0.751± 0.025
0.384± 0.022
1.295± 0.037
1.851± 0.076*
0.596± 0.022
Ethanol + fructose
0.841± 0.018*
0.907± 0.037
0.463± 0.016
2.033± 0.046
2.541± 0.171*
0.648± 0.018
Ethanol + glucose&fructose 1.865± 0.031*
0.673± 0.073
0.305± 0.011
1.308± 0.044
1.810± 0.088*
0.492± 0.024
Values are expressed as the mean ± SD of n determinations (n=5 rabbits per group); *: Significantly different from the control-treated value (p< 0.05);
TAG: Triacylglycerol; T.chol: Total cholesterol
Table 4:Changes in feed consumption and body weight measures induced by glucose ad fructose co-administration to ethanol-treated albino rabbits
Duration of Treatment (wk)
-------------------------------------------------------------------------------------------------------------------------Treatment
0
5
10
15
Changes in feed consumption (g/rabbit/wk)
Control
362±16
359±13
357±14
Ethanol
366±18
357±16
317±21*
Ethanol + Glucose
364±17
361±15
324±19*
Ethanol + Fructose
365±18
358±21
327±20*
Ethanol + Glucose & Fructose
368±22
363±19
336±17*
Changes in body weight measurements (kg)
Control
1.45±0.12
1.47±0.48
1.46±0.49
1.44±0.43
Ethanol
1.45±0.12
1.43±0.51
1.36±0.62
1.31±0.55**
Ethanol + Glucose
1.45±0.12
1.46±0.42
1.42±0.61
1.38±0.62
Ethanol + Fructose
1.45±0.12
1.48±0.47
1.40±0.58
1.37±0.49
Ethanol + glucose Fructose
1.45±0.12
1.44±0.61
1.39±0.56
1.32±0.56**
Values are expressed as Mean ± SD for n = 7 rabbits per treatment group; *: Significantly different from comparable control value (p<0.05);
**: Significantly different from both comparable control and basal (0 week) values
Fig. 1: Section of liver tissue from the normal saline treated
rabbits showing central vein, surrounding sinusoids,
portal triad, and plates of hepatocytes. (H&E Staining x
40) Section shows features of normal hepatic tissue
Fig. 2 :
ethanol + fructose treatment increased the levels of total
cholesterol the most in all the three tissues. Ethanol +
glucose and fructose administration significantly
(p<0.05) increased liver TAG but lowers TAG level in the
heart (p<0.05). Apart from ethanol + glucose treatment,
the other three treatments produced significant increase
(p<0.05) in liver TAG levels, but all treatments caused
significant changes in heart TAG. The various treatments
had no significant effect on brain TAG.
Section of liver tissue from ethanol-treated rabbits
showing hepatocytes and portal tracts. There is
presence of inflammatory cells in the portal tracts
consisting of lymphocytes (H&E Staining x 40)
Section shows characteristic features of hepatitis
Figure 1, 2, 3, 4 and 5 show the histopathological
sections of the liver for the control, ethanol, ethanol +
glucose, ethanol + fructose and ethanol + glucose and
fructose-treated rabbits.
Figure 2, 4 and 5 show evidence of fatty hepatitis,
while Fig. 3 shows hepatic congestion. Figure 1 shows
normal features.
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Asian J. Med. Sci., 3(1): 1-7, 2011
Fig. 5: Section of liver tissue from the ethanol +glucose and
fructose-treated rabbits showing inflammatory cells and
extension of fibrosis and the inflammation into the
hepatic parenchyma. (H&E Staining x 40). Features
show hepatitis with fibrosis
Fig. 3: Section of liver tissue from the ethanol + glucose-treated
rabbits showing hepatic congestion (H&E Staining x
40). Section shows hepatic congestion
and Anosike, 2004). However, glucose + fructose coadministration increased the degree of BEER stimulated
by fructose alone. While this adds to the accumulating
information on the effects of glucose on fructose
absorption (Truswell et al., 1988), the data also suggest
that the co-administration of glucose and fructose may
improve the metabolic utilization of fructose. Albeit, this
co-administration initiates a progressive increase in blood
(Table 2) and liver TAG (Table 3), and induces a
measure of structural damage (fatty hepatitis) to
hepatocytes (Fig. 5).
Many of the consequences of excessive alcohol
consumption are the result of redox changes occurring in
the course of the metabolism of ethanol. Oxidation of
ethanol to acetaldehyde and subsequently to acetate leads
to an increase in the (NADH)/(NAD+) ratio which
contributes to the accumulation of fat (TAG) in the liver
of alcoholics (Lieber, 1994). An increased
(NADH)/(NAD+) ratio may also stimulate the
mitochondrial respiratory chain. The resulting increase in
election flow along the respiratory chain generates
reactive oxygen species that have been implicated in
ethanol-associated cell injury (Bailey et al., 1999). The
findings in the ethanol-treated rabbits are consistent with
these hypotheses. It follows that interventions to decrease
the concentration of NADH during metabolism of ethanol
would theoretically reduce the degree of ethanol-induced
biochemical changes and associated organic lesions
(Bailey and Cunningham, 1998). In support of this theory,
fructose in part alleviates the ethanol-induced redox shift
and increases the rate of disappearance of ethanol from
blood (Berman et al., 2003; Rawat, 1977), an effect which
is thought to be due to facilitation of intra-mitochondrial
re-oxidation of NADH (Crownover et al., 1986). Thus, it
makes sense to say that the use of fructose to stimulate
Fig. 4: Section of liver tissue from the ethanol + fructosetreated rabbits showing the presence of inflammatory
cells and fibrosis. (H&E Staining x 40). Features show
chronic non-specific hepatitis
Table 4 shows the changes in feed consumption and body
weight measures induced by glucose and fructose coadministration to ethanol-treated albino rabbits. Data
indicate that ethanol administration significantly (p<0.05)
reduced the amount of feed consumed and body weight at
the 15th week of administration. Glucose and fructose
co-administration had similar effect on ethanol-treated
rabbits. The administration of either glucose or fructose
reduced (p<0.05) feed consumed but no significant effect
(p>0.05) on body weight.
DISCUSSION
Available data (Table 1) indicate that fructose
stimulated BEER in rabbits and this supports previous
findings among humans (Berman et al., 2003; Onyesom
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Asian J. Med. Sci., 3(1): 1-7, 2011
Berman, P.A.M., I. Baumgarten and D.L. Viljoen, 2003.
Effect of oral fructose on ethanol elimination from
the bloodstream. South Afr. J. Sci., 99: 47-50.
Bucher, T.H. and H. Redetzki, 1951. Eine spezifische
photometrische Bestimmung von Athylalkohol
auf fermentativen Wege. Klin. Wochenschr., 26:
615-620.
Burstein, M. and R. Mortin, 1969. Quantitative
determination of HDL-cholesterol using the
enzymatic-colorimetric method. Life Sci., 8:
345-347.
Chawla, R., 1999. Practical Clinical Biochemistry:
Methods and Interpretations. 2nd Edn., Jaypee
Brothers Medical Publisher Ltd., New Delhi.
Crownover, B.P., J. LaDine, B. Bradford, E. Glassman,
D. Forman, H. Schneider and R.G. Thurman, 1986.
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Ethanol modulates apolipo protein B mRNA editing
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Lieber, C.S., 1994. Alcohol and the liver-1994 update.
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Mascord, D., J. Smith, G.A. Starmer and J.B. Whitfield,
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Onyesom, I. and E.O. Anosike, 2004. Oral fructoseinduced disposition of blood ethanol and associated
changes in plasma urate. Afr. J. Drug Alcohol Stud.,
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ethanol elimination from bloodstream would also reduce
the levels of some biochemical analytes and possibility of
cell injury.
In contrast, however, this study reveals that
ethanol + fructose administration further increased blood
(Table 2), liver, brain and heart TAG levels (Table 3), and
induced fatty hepatitis (Fig. 4). The co-administration of
ethanol + fructose and glucose produced the highest
increase in blood and hepatic TAG at the end of the 15
week treatment period, and these may have contributed to
the fatty (fibrotic) hepatitis observed (Fig. 5).
In general, it follows that the further increase in blood
TAG induced by fructose in the presence of ethanol may
not be wholly due to the (NADH)/(NAD+) redox shift.
Fructose metabolism bypasses some of the regulatory
steps in glycolysis and this causes progression of
glycolysis in the liver in an uncontrolled manner
(Hallfrisch, 1990). Uncontrolled glycolysis leads to
immediate increase in pyruvate and lactate levels and this
activates pyruvate dehydrogenase enzyme which produces
a shift from fatty acid oxidation for energy to fatty acid
esterification in preparation for release from the liver in
the form of VLDL, promoting fat (TAG) formation and
VLDL production and secretion rather than glycogen
formation.
One of the essential parameters in ethanol feeding
studies is to monitor the general healthiness of the
animals, which is most often reflected in their body
weight and feed consumption. Ethanol + fructose and
glucose co-administration, significantly reduced the
amount of feed consumed and body weight at the 15th
week of exposure. These physical measurements
authenticate the biochemical data and histopathological
state, and altogether, they indicate unhealthy condition.
The idea of adding glucose to the administration of
fructose in rabbits fed with ethanol may not be a
wholesome practice because it did not produce the
positive effects of healthiness of the animals as evidenced
by the reduced feed intake and weight lose. In addition, it
increased the liver tissue levels of TAG, thus, enhancing
the risk of fatty hepatitis.
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